Fungal Denitrification Research Articles

Mapping N2O production pathways in estuarine and coastal sediments through coupled 15N tracing and N2O isotopocules analysis

Estuarine and coastal zones are important sources of atmospheric nitrous oxide (N2O). Previous studies have explored the overarching roles of nitrification and denitrification in N2O production from these critical zones, yet the specific productions of various pathways within these processes remain elusive. Using dual 15N tracers (15NH4+ and 15NO3-) coupled with N2O isotopocules analysis, we quantified the contributions of multiple nitrification (both autotrophic and heterotrophic) and denitrification (bacterial, fungal, and chemical) pathways to N2O production in estuarine and coastal sediments. Furthermore, we leveraged microecology theory to dissect the biogeochemical mechanisms that govern the spatiotemporal dynamics of these pathways. Results showed that denitrification was the dominant process, accounting for over 70 % of N2O production. The remainder was attributed to autotrophic nitrification (5.80–23.92 %) and heterotrophic nitrification (1.07–7.10 %). Isotopocules analyses further showed that fungal denitrification was the primary source at freshwater sites (40.47–49.99 %), whereas bacterial denitrification prevailed at high-salinity sites (51.37–52.55 %). Moreover, these processes displayed contrasting spatial variability along the estuarine salinity gradient. Mechanistic investigation informed by microecology theory demonstrated that distinct assembly processes governed bacterial and fungal communities across this gradient, resulting in divergent ecological adaptation strategies of bacterial and fungal denitrifiers and, consequently, the contrasting contribution patterns of bacterial and fungal denitrification. Collectively, our work presents a comprehensive and refined picture of N2O sources and dynamics, offering essential insights for improving predictive models and formulating effective mitigation strategies targeting N2O emissions from estuarine and coastal zones.

Effects of inhibitors and slit incorporation on NH3 and N2O emission processes after urea application

The use of urea fertilizers in agriculture is associated with many negative environmental impacts and is a source of ammonia (NH3) and nitrous oxide (N2O) emissions. Such losses from urea fertilizer can be avoided by different mitigation techniques. Three different mitigation principles, urease inhibitor (N-(2-Nitrophenyl) phosphoric triamide, 2-NPT) (UI) alone and urease inhibitor in combination with nitrification inhibitors (N-[3(5)-methyl-1 H-pyrazol-1-yl) methyl] acetamide, MPA) (NI) and closed slit incorporation of urea fertilizer into the soil, were compared on a sandy loam soil at a soil water level of 70 % water-holding capacity. An in vitro microcosm approach with open dynamic incubation chambers was used to monitor NH3 emissions over two weeks with NH3 sampling by washing bottles. N2O emissions were studied over ten weeks in slow throughflow mesocosms with continuous gas chromatographic (GC) measurements. To get insights into N2O production and consumption processes, gas samples were taken after six weeks and N2O isotopocules were analyzed by isotope ratio mass spectrometry (IRMS). Slit injection showed the greatest effect on NH3 emission reduction by 79.6 % (40.6 % by UI, and 46.7 % by UINI) compared to surface applied urea. Minor pollution swapping to N2O was observed at the beginning of the trial due to incorporation but not in the cumulative emissions over the entire incubation time. The reduction effect of UINI on N2O emissions decreased over time with no cumulative emission reduction at the end of experimentation. N2O isotopocules confirmed the high contribution of nitrification to N2O production. In contrast and bacterial denitrification, nitrifier denitrification and fungal denitrification were involved on a much lower level and N2O reduction to N2 was not pronounced. All NH3 mitigation measurements where effective to decrease NH3 emissions while their effects on N2O emission varied over time. Factors as crop N uptake and rainfall would further modify the overall effect on N2O emissions and need to be considered for final pollution swapping assessment. Further research on the impact of NI on non-target microbial communities is warranted to elucidate potential environmental consequences and long-term efficacy of inhibitor compounds.

Partitioning denitrification pathways in N2O emissions from re-flooded dry paddy soils

In flooded paddy fields, peak greenhouse gas nitrous oxide (N2O) emission after rewetting the dry soils is widely recognised. However, the relative contribution of biotic and abiotic factors to this emission remains uncertain. In this study, we used the isotope technique (δ18O and δ15NSP) and molecular-based microbial analysis in an anoxic incubation experiment to evaluate the contributions of bacterial, fungal, and chemical denitrification to N2O emissions. We collected eight representative paddy soils across southern China for an incubation experiment. Results show that during the 10-day incubation period, the net N2O emissions were mainly produced by fungal denitrification, which accounted for 58–77% in six of the eight investigated flooded paddy soils. In contrast, bacterial denitrification contributed 6–15% of the net N2O emissions. Moreover, around 11–35% of the total N2O emissions were derived from chemical denitrification in all soil types. Variation partitioning analysis (VPA) and principal component analysis (PCA) demonstrated that initial soil organic carbon (OC) concentrations were the primary regulator of N2O source patterns. Soils with relatively lower OC concentration (7–15 mg g−1) tend to be dominated by fungal denitrification, which accounted for the net N2O production at the end of the incubation period. Overall, these findings highlight the dominance of the fungal denitrification pathway for N2O production in flooded paddy soils, which predominates in soils with relatively lower OC content. This suggests that fungal contribution should be considered when optimizing agricultural management system timing to control N2O emissions in flooded paddy soil ecosystems, and for the relevant establishment of predictive numerical models in the future.

Effects of Long-term Biochar Addition on Denitrification N2O Emissions from Bacteria and Fungi in Paddy Soil

Denitrification driven by bacteria and fungi is the main source of nitrous oxide （N2O） emissions from paddy soil. It is generally believed that biochar reduces N2O emissions by influencing the bacterial denitrification process, but the relevant mechanism of its impact on fungal denitrification is still unclear. In this study, the long-term straw carbonization returning experimental field in Changshu Agricultural Ecological Experimental Base of the Chinese Academy of Sciences was taken as the object. Through indoor anaerobic culture and molecular biology technology, the relative contributions of bacteria and fungi to denitrifying N2O production in paddy soil and the related microorganism mechanism were studied under different long-term biochar application amounts （blank, 2.25 t·hm-2, and 22.5 t·hm-2, respectively, expressed by BC0, BC1, and BC10）. The results showed that compared with that in BC0, biochar treatment significantly reduced N2O emission rate, denitrification potential, and cumulative N2O emissions, and the contribution of bacterial denitrification was greater than that of fungal denitrification in all three treatments. Among them, the relative contribution rate of bacterial denitrification in BC10 （62.9%） was significantly increased compared to BC0 （50.8%）, whereas the relative contribution rate of fungal denitrification in BC10 （37.1%） was significantly lower than that in BC0 （49.2%）. The application of biochar significantly increased the abundance of bacterial denitrification functional genes （nirK, nirS, and nosZ） but reduced the abundance of fungal nirK genes. The contribution rate of fungal denitrification was significantly positively correlated with the N2O emission rate and negatively correlated with soil pH, TN, SOM, and DOC. Biochar may have inhibited the growth of denitrifying fungi by increasing pH and carbon and nitrogen content, reducing the abundance of related functional genes, thereby weakening the reduction ability of NO to N2O during fungal denitrification process. This significantly reduces the contribution rate of N2O production during the fungal denitrification process and the denitrification N2O emissions from paddy soil. This study helps to broaden our understanding of the denitrification process in paddy soil and provides a theoretical basis for further regulating fungal denitrification N2O emissions.

Acidification Offset Warming-Induced Increase in N2O Production in Estuarine and Coastal Sediments.

Global warming and acidification, induced by a substantial increase in anthropogenic CO2 emissions, are expected to have profound impacts on biogeochemical cycles. However, underlying mechanisms of nitrous oxide (N2O) production in estuarine and coastal sediments remain rarely constrained under warming and acidification. Here, the responses of sediment N2O production pathways to warming and acidification were examined using a series of anoxic incubation experiments. Denitrification and N2O production were largely stimulated by the warming, while N2O production decreased under the acidification as well as the denitrification rate and electron transfer efficiency. Compared to warming alone, the combination of warming and acidification decreased N2O production by 26 ± 4%, which was mainly attributed to the decline of the N2O yield by fungal denitrification. Fungal denitrification was mainly responsible for N2O production under the warming condition, while bacterial denitrification predominated N2O production under the acidification condition. The reduced site preference of N2O under acidification reflects that the dominant pathways of N2O production were likely shifted from fungal to bacterial denitrification. In addition, acidification decreased the diversity and abundance of nirS-type denitrifiers, which were the keystone taxa mediating the low N2O production. Collectively, acidification can decrease sediment N2O yield through shifting the responsible production pathways, partly counteracting the warming-induced increase in N2O emissions, further reducing the positive climate warming feedback loop.

Nitrate reduction coupling with As(III) oxidation in neutral As-contaminated paddy soil preserves nitrogen, reduces N2O emissions and alleviates As toxicity

In arsenic (As)-contaminated paddy soil, microbial-driven nitrate (NO3−) reduction coupled with arsenite (As(III)) oxidation can reduce As toxicity, but the whereabouts of NO3− remain unclear. In this study, the experiments were established using selective streptomycin (STP) and cyclohexylamine to inhibit bacterial and fungal functional responses, respectively, and metagenomic sequencing techniques were used to explain the biological mechanisms of NO3− reduction coupled with As(III) oxidation in neutral As-contaminated paddy soil. The results indicated that fungal denitrification resulted in stronger nitrous oxide (N2O) emissions (321.6 μg kg−1) than bacterial denitrification (175.9 μg kg−1) in neutral As-contaminated paddy soil, but NO3− reduction coupled with As(III) oxidation reduced the N2O emissions. Only adding STP led to ammonium (NH4+) generation (17.7 mg kg−1), and simultaneously more NH4+ appeared in NO3− reduction coupled with As(III) oxidation; this may be because it improved the electron transfer efficiency by 18.2 %. Achromobacter was involved in denitrification coupled with As(III) oxidation. Burkholderiales was responsible for NO3− reduction to NH4+ coupled with As(III) oxidation. This study provided a theoretical basis for NO3− reduction coupled with As(III) oxidation reducing N2O emissions, promoting the reduction of NO3− to NH4+, and reducing As toxicity in paddy soil.

Unravelling spatiotemporal N2O dynamics in an urbanized estuary system using natural abundance isotopes

Estuaries are strong sources of N2O to the atmosphere; yet we still lack insights into the impact of their biogeochemical dynamics on the emissions of this powerful greenhouse gas. Here, we investigated the spatiotemporal dynamics of the N cycle in an estuary with a focus on the emission mechanisms and pathways of N2O. By coupling N2O isotopocule analysis and substrate NO3− isotope analysis, we found that nutrient availability, oxygen level, salinity gradient and temperature variation were major drivers of the N2O emissions from the Scheldt Estuary. In winter, lower temperature and higher O2 concentration diminished denitrification rates and reduction of N2O to N2, while both were enhanced in warmer summer, causing higher fraction of reduced N2O. As a result, we found comparable N2O fluxes and dissolved concentrations between the two seasons. Decrease in salinity level and increase in NO3− concentration accelerated N2O production when moving upstream of the estuary where more urbanization and higher NO3− from wastewater discharges were found. However, these drivers had no significant effect on the fraction of N2O derived by either denitrification or nitrification and/or fungal denitrification since the fractional proportion of these pathways showed no spatiotemporal variations, remaining around 89 % and 11 %, respectively. These findings challenge the conventional notion that N2O fluxes are generally higher in summer because of higher denitrification rates while confirming that denitrification is the most important pathway of N2O production in the estuaries. Furthermore, our study highlight the importance of combining various isotope analyses to gain in-depth understanding about N2O emission pathways and N cycling in dynamic systems like estuaries.

Stabilization of a Heme-HNO Model Complex Using a Bulky Bis-Picket Fence Porphyrin and Reactivity Studies with NO.

Nitroxyl, HNO/NO-, the one-electron reduced form of NO, is suggested to take part in distinct signaling pathways in mammals and is also a key intermediate in various heme-catalyzed NOx interconversions in the nitrogen cycle. Cytochrome P450nor (Cyt P450nor) is a heme-containing enzyme that performs NO reduction to N2O in fungal denitrification. The reactive intermediate in this enzyme, termed "Intermediate I", is proposed to be an Fe-NHO/Fe-NHOH type species, but it is difficult to study its electronic structure and exact protonation state due to its instability. Here, we utilize a bulky bis-picket fence porphyrin to obtain the first stable heme-HNO model complex, [Fe(3,5-Me-BAFP)(MI)(NHO)], as a model for Intermediate I, and more generally HNO adducts of heme proteins. Due to the steric hindrance of the bis-picket fence porphyrin, [Fe(3,5-Me-BAFP)(MI)(NHO)] is stable (τ1/2 = 56 min at -30 °C), can be isolated as a solid, and is available for thorough spectroscopic characterization. In particular, we were able to solve a conundrum in the literature and provide the first full vibrational characterization of a heme-HNO complex using IR and nuclear resonance vibrational spectroscopy (NRVS). Reactivity studies of [Fe(3,5-Me-BAFP)(MI)(NHO)] with NO gas show a 91 ± 10% yield for N2O formation, demonstrating that heme-HNO complexes are catalytically competent intermediates for NO reduction to N2O in Cyt P450nor. The implications of these results for the mechanism of Cyt P450nor are further discussed.

Responses of soil bacterial and fungal denitrification and associated N2O emissions to organochloride pesticide

The extensive application of organochloride pesticides in agriculture has raised concerns about their potential negative impacts on soil microbial denitrification and associated N2O emissions. However, most studies have primarily focused on bacteria, and the contribution of fungi to N2O emissions and their response to organochloride pesticides have often been overlooked. In this study, 15N tracing combined with the respiration inhibition method was applied to examine the impacts of chlorothalonil on both fungal and bacterial denitrification. The results demonstrated that fungal N2O emissions dominated in the absence of chlorothalonil, accounting for 73 % of total N2O emissions. Chlorothalonil inhibited fungal and bacterial denitrification via different mechanisms and altered the main pathways of soil N2O emissions. Amplicon sequencing analyses indicated that chlorothalonil significantly reduced the abundances of N2O-producing fungi owing to its fungicidal effect and fungal N2O emissions significantly dropped. Molecular biological analyses revealed that chlorothalonil induced lower electron generation, transport, and consumption efficiencies, which led to the inhibition of denitrifying enzymes in bacteria. Bacterial N2O emissions dramatically increased and became the dominant source. These findings provide insights into the mechanisms by which N2O emissions from fungal and bacterial denitrification are influenced by chlorothalonil.

Soil microbial communities as potential regulators of N2O sources in highly acidic soils

• Soil pH was a key driver of N2O emission and sources in acidic soils. • N2O emission was significantly positively associated with the ratio of ITS to 16S. • N2O was significantly correlated with bacterial and fungal community composition. • Fungi contributed to N2O in highly acidic tea plantations and vegetable fields. Acidic soil is a main source of global nitrous oxide (N2O) emissions. However, the mechanism behind the high N2O emissions from acidic soils remains a knowledge gap. The objective of this microcosm incubation study was to pin-point the microbial mechanisms involved in N2O production processes in acidic soils. For that purpose, the isotopic signatures and microbial community structure and composition of four soil samples were examined. The results showed that highly acid soils (pH = 3.51) emitted 89 times more N2O than alkaline soils (pH = 7.95) under the same nitrogen (N) inputs. Fungal denitrification caused high N2O emissions in acidic soils. ITS to 16S abundance ratio was positively correlated with cumulative N2O emissions from the tested soils. The highly acid soils (pH < 4.5) showed greater fungal nirK gene abundance and lower abundance of AOA-amoA, AOB-amoA, nirK, nosZ I and nosZ II genes. The unclassified Aspergillaceae fungi (63.65%) dominated the highly acidic soils and was the most strongly correlated genus with N2O emissions. These findings highlight that soil microbial community structures, denitrifying fungi in particular, shaped by low pH (pH < 4.5) lead to high N2O emissions from acidic soils.

In situ 15 N-N2 O site preference and O2 concentration dynamics disclose the complexity of N2 O production processes in agricultural soil.

Arable soil continues to be the dominant anthropogenic source of nitrous oxide (N2 O) emissions owing to application of nitrogen (N) fertilizers and manures across the world. Using laboratory and in situ studies to elucidate the key factors controlling soil N2 O emissions remains challenging due to the potential importance of multiple complex processes. We examined soil surface N2 O fluxes in an arable soil, combined with in situ high-frequency measurements of soil matrix oxygen (O2 ) and N2 O concentrations, in situ 15 N labeling, and N2 O 15 N site preference (SP). The in situ O2 concentration and further microcosm visualized spatiotemporal distribution of O2 both suggested that O2 dynamics were the proximal determining factor to matrix N2 O concentration and fluxes due to quick O2 depletion after N fertilization. Further SP analysis and in situ 15 N labeling experiment revealed that the main source for N2 O emissions was bacterial denitrification during the hot-wet summer with lower soil O2 concentration, while nitrification or fungal denitrification contributed about 50.0% to total emissions during the cold-dry winter with higher soil O2 concentration. The robust positive correlation between O2 concentration and SP values underpinned that the O2 dynamics were the key factor to differentiate the composite processes of N2 O production in in situ structured soil. Our findings deciphered the complexity of N2 O production processes in real field conditions, and suggest that O2 dynamics rather than stimulation of functional gene abundances play a key role in controlling soil N2 O production processes in undisturbed structure soils. Our results help to develop targeted N2 O mitigation measures and to improve process models for constraining global N2 O budget.

Bacillus velezensis SQR9 inhibition to fungal denitrification responsible for decreased N2O emissions from acidic soils

Tropical and subtropical acidic soils are hotspots of global terrestrial nitrous oxide (N2O) emissions, with N2O produced primarily through denitrification. Plant growth-promoting microbes (PGPMs) may effectively mitigate soil N2O emissions from acidic soils, achieved through differential responses of bacterial and fungal denitrification to PGPMs. To test this hypothesis, we conducted a pot experiment and the associated laboratory trials to gain the underlying insights into the PGPM Bacillus velezensis strain SQR9 effects on N2O emissions from acidic soils. SQR9 inoculation significantly reduced soil N2O emissions by 22.6–33.5 %, dependent on inoculation dose, and increased the bacterial AOB, nirK and nosZ genes abundance, facilitating the reduction of N2O to N2 in denitrification. The relative contribution of fungi to the soil denitrification rate was 58.4–77.1 %, suggesting that the N2O emissions derived mainly from fungal denitrification. The SQR9 inoculation significantly inhibited the fungal denitrification and down-regulated fungal nirK gene transcript, dependent on the SQR9 sfp gene, which was necessary for secondary metabolite synthesis. Therefore, our study provides new evidence that decreased N2O emissions from acidic soils can be due to fungal denitrification inhibited by PGPM SQR9 inoculation.

Identification of Fungal and Bacterial Denitrification Inhibitors Targeting Copper Nitrite Reductase.

The usage of nitrification inhibitors is one of the strategies that reduce or slow down the denitrification process to prevent nitrogen loss to the atmosphere in the form of N2O. Directly targeting microbial denitrification could be one of the mitigation strategies; however, until now little efforts have been devoted toward the development of denitrification inhibitors. Here, we have identified small-molecule inhibitors of one of the proteins involved in the fungal denitrification pathway. Specifically, virtual screening was employed to identify the inhibitors of copper-containing nitrite reductase (FoNirK) of the filamentous fungus Fusarium oxysporum. Three series of chemical compounds were identified, out of which compounds belonging to two chemical scaffolds inhibited FoNirK enzymatic activity in low micromolar ranges. Several compounds also displayed moderate inhibition of fungal denitrification activity in vivo. Evaluation of in vitro activity against NirK from denitrifying bacterium Achromobacter xylosoxidans (AxNirK) and in vivo bacterial denitrification revealed a similar inhibitory profile.

Hot moment of N2O emissions in seasonally frozen peatlands.

Since the start of the Anthropocene, northern seasonally frozen peatlands have been warming at a rate of 0.6 °C per decade, twice that of the Earth's average rate, thereby triggering increased nitrogen mineralization with subsequent potentially large losses of nitrous oxide (N2O) to the atmosphere. Here we provide evidence that seasonally frozen peatlands are important N2O emission sources in the Northern Hemisphere and the thawing periods are the hot moment of annual N2O emissions. The flux during the hot moment of thawing in spring was 1.20 ± 0.82 mg N2O m-2 d-1, significantly higher than that during the other periods (freezing, -0.12 ± 0.02 mg N2O m-2 d-1; frozen, 0.04 ± 0.04 mg N2O m-2 d-1; thawed, 0.09 ± 0.01 mg N2O m-2 d-1) or observed for other ecosystems at the same latitude in previous studies. The observed emission flux is even higher than those of tropical forests, the World's largest natural terrestrial N2O source. Furthermore, based on soil incubation with 15N and 18O isotope tracing and differential inhibitors, heterotrophic bacterial and fungal denitrification was revealed as the main source of N2O in peatland profiles (0-200 cm). Metagenomic, metatranscriptomic, and qPCR assays further revealed that seasonally frozen peatlands have high N2O emission potential, but thawing significantly stimulates expression of genes encoding N2O-producing protein complexes (hydroxylamine dehydrogenase (hao) and nitric oxide reductase (nor)), resulting in high N2O emissions during spring. This hot moment converts seasonally frozen peatlands into an important N2O emission source when it is otherwise a sink. Extrapolation of our data to all northern peatland areas reveals that the hot moment emissions could amount to approximately 0.17 Tg of N2O yr-1. However, these N2O emissions are still not routinely included in Earth system models and global IPCC assessments.

Fungi contribute more to N2O emissions than bacteria in two paddy soils with different textures

Both bacteria and fungi contribute to nitrous oxide (N2O) emissions through denitrification in soils; however their contributions to N2O emissions in paddy soil with different soil textures are unclear. In this study, N2O emission flux and production potential were determined using the substrate-induced respiration (SIR) inhibition method with or without acetylene addition. Fungi contributed more to N2O emission flux than bacteria in loam and clay soils, and their contribution to N2O production potential was higher than that of bacteria in loam soil. Moreover, the contributions of fungi and bacteria to N2O emission flux and production potential were higher in clay soil than in loam soil. Thus, fungi play a significant role in N2O emissions from paddy soils, which was highly dependent on soil texture. Therefore, the role of fungal denitrification in N2O emission flux in paddy soils should be investigated in future research.

Increased Nitrogen Loading Facilitates Nitrous Oxide Production through Fungal and Chemodenitrification in Estuarine and Coastal Sediments.

Estuarine and coastal environments are assumed to contribute to nitrous oxide (N2O) emissions under increasing nitrogen loading. However, isotopic and molecular mechanisms underlying N2O production pathways under elevated nitrogen concentration remain poorly understood. Here we used microbial inhibition, isotope mass balance, and molecular approaches to investigate N2O production mechanisms in estuarine and coastal sediments through a series of anoxic incubations. Site preference of the N2O molecule increased due to increasing nitrate concentration, suggesting the changes in N2O production pathways. Enhanced N2O production under high nitrate concentration was not mediated by bacterial denitrification, but instead was mainly regulated by fungal denitrification. Elevated nitrate concentration increased the contribution of fungal denitrification to N2O production by 11-25%, whereas it decreased bacterial N2O production by 16-33%. Chemodenitrification was also enhanced by high nitrate concentration, contributing to 13-28% of N2O production. Elevated nitrate concentration significantly mediated nirK-type denitrifiers structure and abundance, which are the keystone taxa driving N2O production. Collectively, these results suggest that increasing nitrate concentration can shift N2O production pathways from bacterial to fungal and chemodenitrification, which are mainly responsible for the enhanced N2O production and have widespread implications for N2O projections under ongoing nitrogen pollution in estuarine and coastal ecosystems.

Nitrous oxide production and isotopomer composition by fungi isolated from salt marsh sediments

The emissions of nitrous oxide (N2O), a potent greenhouse gas and ozone-depleting agent, have been steadily increasing from coastal environments, such as salt marsh sediments, as a result of anthropogenic nutrient loading. Biotic processes, including nitrification and denitrification, are the largest sources of N2O from salt marsh sediments. While it is assumed that the bulk of N2O from salt marsh sediment is produced by nitrification and bacterial denitrification, recent reports suggest fungal denitrification may contribute significantly. In this study, four fungi capable of growth under sulfidic conditions were isolated from salt marsh sediments in North Inlet, South Carolina, USA. Fungal species included Purpureocillium lilacinum, Trichoderma harzianum, Trichoderma virens, and Rhodotorula glutinis, as determined by sequencing the18S and 28S rRNA genes. The isotopomer signatures of N2O produced by these fungi were measured using isotope ratio mass spectrometry, which can be used to estimate the contribution of different sources of N2O. Up to 22.8% of nitrite provided in growth media was converted to N2O by fungal strains isolated from salt marsh sediments. The site preference (SP) of N2O produced by salt marsh sediment fungi ranged from 7.5 ± 1.6‰ to 33.4 ± 1.2‰. These values are lower than the SP of N2O from the model fungal denitrifier Fusarium oxysporum (37.1 ± 2.5‰), which is the SP typically used as an endmember in isotope mass balance considerations. The N2O SP values we measured expand the range of N2O SP used for isotope mass balances calculations to determine the relative contribution of fungi to N2O production in salt marsh sediments.

Denitrification and N2O Emission in Estuarine Sediments in Response to Ocean Acidification: From Process to Mechanism.

Global estuarine ecosystems are experiencing severe nitrogen pollution and ocean acidification (OA) simultaneously. Sedimentary denitrification is an important way of reactive nitrogen removal but at the same time leads to the emission of large amounts of nitrous oxide (N2O), a potent greenhouse gas. It is known that OA in estuarine regions could impact denitrification and N2O production; however, the underlying mechanism is still underexplored. Here, sediment incubation and pure culture experiments were conducted to explore the OA impacts on microbial denitrification and the associated N2O emissions in estuarine sediments. Under neutral (in situ) conditions, fungal N2O emission dominated in the sediment, while the bacterial and fungal sources had a similar role under acidification. This indicated that acidification decreased the sedimentary fungal denitrification and likely inhibited the activity of fungal denitrifiers. To explore molecular mechanisms, a denitrifying fungal strain of Penicillium janthinellum was isolated from the sediments. By using deuterium-labeled single-cell Raman spectroscopy and isobaric tags for relative and absolute quantitation proteomics, we found that acidification inhibited electron transfers in P. janthinellum and downregulated expressions of the proteins related to energy production and conservation. Two collaborative pathways of energy generation in the P. janthinellum were further revealed, that is, aerobic oxidative phosphorylation and TCA cycle and anoxic pyruvate fermentation. This indicated a distinct energy supply strategy from bacterial denitrification. Our study provides insights into fungi-mediated nitrogen cycle in acidifying aquatic ecosystems.

Minimizing tillage modifies fungal denitrifier communities, increases denitrification rates and enhances the genetic potential for fungal, relative to bacterial, denitrification

Nitrous oxide (N2O) emissions from arable soils are predominantly caused by denitrifying microbes, of which fungal denitrifiers are of particular interest, as fungi, in contrast to bacteria, terminate denitrification with N2O. Reduced tillage has been shown to increase gaseous nitrogen losses from soil, but knowledge of how varying tillage regimes and associated soil physical and chemical alterations affect fungal denitrifiers is limited. Based on results from a long-term (>40 years) tillage experiment, we show that non-inversion tillage resulted in increased potential denitrification activity in the upper soil layers, compared to annual or occasional (every 4–5 years) conventional inversion tillage. Using sequence-corrected abundance of the fungal nirK gene, we further identified an increased genetic potential for fungal denitrification, compared to that caused by bacteria, with decreasing tillage intensity. Differences in the composition and diversity of the fungal nirK community imply that different tillage regimes select for distinct fungal denitrifiers with differing functional capabilities and lifestyles, predominantly by altering carbon and nitrogen related niches. Our findings suggest that the creation of organic hotspots through stratification by non-inversion tillage increases the diversity and abundance of fungal denitrifier communities and modifies their composition, and thus their overall relevance for N2O production by denitrification, in arable soils.

Poor recovery of fungal denitrification limits nitrogen removal capacity in a constructed Gulf Coast marsh

Widespread degradation and destruction of coastal wetlands over the last century have spurred on the practice of creating salt marshes to mitigate losses of wetland area and ecosystem function. Constructed marshes can quickly recover plant biomass, but biogeochemical functions, such as nitrogen removal capacity through denitrification, can take decades to centuries to recover. One potential mechanism for this uncoupling of structural and functional recovery is that an impaired microbial community subsequently impedes the nitrogen removal capacity of constructed marshes. While both bacteria and fungi can contribute to nitrogen removal via denitrification, little is known about fungal denitrification in wetlands, even though it has the potential to contribute to climate change via the production of the greenhouse gas nitrous oxide. Here, we measured fungal and bacterial denitrification potential rates and fungal biomass in sediments collected from a 33-year-old constructed marsh and a reference natural marsh to assess both a) the relative contribution of each group to total nitrogen removal and b) whether fungal biomass accrual is a driver of functional recovery in salt marshes. To assess the relative contributions of fungi and bacteria to denitrification, we added selective inhibitors (antifungal, antibacterial, or both) to sediments from each marsh and measured potential fungal and bacterial denitrification rates. We also measured sediment ergosterol concentrations seasonally as a proxy for fungal biomass. In the absence of inhibitors, denitrification potential rates in the constructed marsh were three times lower than in natural marsh sediment. Further, denitrification rates in the constructed marsh remained similarly low regardless of increasing inhibitor concentration, whereas denitrification rates in the natural marsh declined with increasing inhibitor concentration. When either inhibitor was added alone, denitrification was stimulated in the natural marsh, but was suppressed in the constructed marsh, suggesting an apparent competitive release, where the reduction of either bacterial or fungal competition for substrate thereby allowed the remaining competitor greater access to the substrate, in the natural marsh. We observed that compared to the constructed marsh, fungi in the natural marsh contributed ∼30% more (50% vs. 21%) to denitrification, and that fungal biomass was generally higher. Collectively, these data suggest that 3 decades post construction, denitrification rates and the contribution of fungal denitrification to total denitrification are lower in the constructed marsh than the natural marsh. These data also suggest that fungal biomass may limit the recovery of nitrogen removal in constructed marshes and that fungal denitrification can be an important pathway of N removal in salt marsh ecosystems.